PROJECT SUMMARY Despite the substantial impact pre-mRNA splicing has on gene expression outcomes, little is known about how the spliceosome itself is modified and regulated during cellular reprogramming. Innate immune cells like macrophages reprogram gene expression when they sense a ?danger signal,? such as a pathogen, organelle damage, or chemical signal, to combat the detected threat. While changes that occur transcriptionally during macrophage activation are well characterized, almost nothing is known about how pre- mRNA splicing is regulated following immune stimuli. The long-term goal of this project is to uncover how macrophage activation modifies the spliceosome and to connect these changes with innate immune gene expression outcomes. The spliceosome is a complex and dynamic macromolecular machine. Its ability to recognize introns and catalyze their removal relies on numerous RNA binding proteins that recognize specific sequences in exons and introns to ?read? the splicing code. The central hypothesis of this proposal is that during macrophage activation, post-translational modification of splicing factors directs assembly of a specialized spliceosome characterized by a distinct cohort of protein-protein interactions that promotes the innate immune gene expression program. In support of this model, phosphoproteomic experiments reveal that 30+ splicing factors, many with known regulatory roles, are phosphorylated or dephosphorylated at specific serine residues following lipopolysaccharide (LPS)-dependent activation of macrophages. Experiments interrogating one such factor, hnRNP M, show that LPS treatment triggers dephosphorylation concomitant with its redistribution in the nucleus. Loss of hnRNP M by shRNA-mediated knockdown in macrophages alters alternative splicing of a number of pre-mRNAs and leads to hyper-induction of important innate immune transcripts, including the potent inflammatory mediator IL-6 and the key viral restriction factor Mx1. This proposal expands upon these observations, looking globally at changes to the spliceosome following macrophage activation. It will combine high-throughput approaches, including affinity purification-mass spectrometry, phosphoproteomics, RNA-seq, and RNA CLIP-seq with targeted genetic and biochemical experiments to implicate specific splicing factors in driving innate immune gene expression changes. This research program will fill key gaps in our knowledge of how splicing is regulated following macrophage activation and further our understanding of how the spliceosome reads and interprets the splicing code not only during innate immune activation but also during other cellular reprogramming, including differentiation, stress, starvation, and carcinogenesis.